Efficient production of bioactive polypeptides and peptides is an important goal of the biomedical and biotechnology industries. Bioactive peptides and proteins are used as therapeutic and diagnostic agents in a variety of diseases such as diabetes (insulin), viral infections and leukemia (interferon), diseases of the immune system (interleukins), and red blood cell deficiencies (erythropoietin), to name a few. Additionally, large quantities of proteins and peptides are needed for various industrial applications including, but not limited to, pulp and paper industries, textiles, food industries, personal care and cosmetics industries, sugar refining, wastewater treatment, production of alcoholic beverages, and as catalysts for the generation of new pharmaceuticals.
In biomedical-related fields small peptides are sometimes used as linkers for the attachment of diagnostic and pharmaceutical agents to surfaces (see U.S. Pat. App. Pub. No. 2003/0185870 to Grinstaff et al. and U.S. Pat. No. 6,620,419 to Lintner, K.). In the field of personal care, small peptides have been used to couple benefit agents to body surfaces such as hair, skin, nail, and teeth (U.S. Pat. Nos. 7,220,405; 7,309,482; 7,129,326; 7,585,495 and 7,285,264; U.S. Pat. App. Pub. Nos. 2002/0098524; 2005/0112692; 2005/0226839; 2007/0196305; 2006/0199206; 2007/0065387; 2008/0107614; 2007/0110686; 2008/0280810; 2006/0171885; and 2008/0175798).
Peptides may be prepared by chemical synthesis or isolated from natural sources. However, these methods are often expensive, time consuming, and characterized by limited production capacity. The preferred method of producing large quantities of peptides or proteins is through the fermentation of recombinant microorganisms engineered to express a genetic construct encoding the peptide or protein of interest. However, recombinant microbial peptide production has a number of obstacles to overcome in order to be cost-effective. For example, peptides produced within a recombinant microbial host cell are often degraded by endogenous proteases, which decrease the yield and increase the cost of production. Additionally, microbial production of smaller peptides in high yield may be adversely affected by size and the amino acid composition of the peptide. This is especially evident when the peptide of interest is soluble under typical physiological conditions found within the production host.
One way to mitigate the difficulties associated with recombinantly producing a soluble peptide of interest (POI) is to produce it in an insoluble form that may accumulate within the host cell as an inclusion body. Soluble POIs may be produced as insoluble fusion proteins by coupling at least one peptidic tag that promotes insolubility (i.e., an inclusion body tag or “IBT”) to the peptide of interest. Producing the peptide of interest in the form of inclusion bodies provides a convenient means to isolate the protein from other cellular components.
One of the difficulties associated with recombinant protein production is controlling the costs associated with processing the recombinant biomass to obtain the desired peptide or protein of interest. Processing steps may include harvesting cells by centrifugation (to “spin down”) to recover the cells from the fermentation medium, lysis or homogenization to disrupt the cells to release the peptide, and the application of various separation methods to isolate the fusion polypeptide. Host cell modifications that aid in distinguishing polypeptides comprising POIs would further decrease the cost of POI recovery. Thus, cellular modifications that render any of these steps more rapid and/or easy to perform would be expected to reduce the cost and/or time associated with processing the recombinant host cells.
Altered expression of endogenous genes and/or the introduction of additional expressible genetic constructs may enhance recombinant peptide/protein production. Chen et al. (Biotech Bioengin (2004) 85 (5):463-474) disclose mutations affecting endogenous periplasmic proteases reported to increase recombinant antibody fragment accumulation in the E. coli periplasmic space. Further, although single gene knockout libraries are available for E. coli (Baba, T., et al., (2006) Mol. Syst. Biol. 2: article 2006.0008), down-regulating or disrupting specific genes or combinations of genes in Escherichia species that significantly effect heterologous peptide production and/or downstream processing are not as well known.
U.S. Pat. No. 7,662,587 to Cheng et al. discloses Escherichia host cells comprising a combination of knockout mutations to gcvA (encoding the glycine cleavage enzyme) and spr (encoding a suppressor of prc) that increased the amount of heterologous peptide produced within the modified host cell. U.S. Pat. Appl. Pub. No. 2010/0227361 to Chen et al. discloses a recombinant Escherichia host cell having a knockout mutation to gcvA, a knockout mutation to spr, and at least one mutation to a portion of the endogenous yejM gene.
Centrifugation is often included as at least one step when recovering the peptide of interest from the recombinant biomass. Rendering cells and/or inclusion bodies denser, larger or heavier than typical inclusion bodies could provide for easier and more rapid isolation. Faster flow rate could be used in continuous centrifugation to harvest cells producing denser inclusion bodies and subsequent washes of the inclusion bodies, which would lead to higher throughput in downstream processing.
One way of achieving this is to provide recombinant host cells having at least one modification that increases the buoyant density of the cell or inclusion bodies within the cell when synthesizing heterologous polypeptides and/or a relative increase in the buoyant density of the inclusion bodies formed within the modified host cell. Increases in buoyant density provides a relatively simple means to identify and obtain cells capable of producing increased amounts of insoluble heterologous protein and/or denser inclusion bodies comprising the heterologous protein.
However, the genetic modifications that one can introduce to a recombinant microbial host cell to increase the buoyant density of the cell or the buoyant density of an inclusion body formed within such a cell are not well understood. The problem to be solved is to provide a method to obtain recombinant host cells having at least one modification that enables more efficient isolation of recombinant polypeptides, including fusion polypeptides comprising the polypeptide of interest.
Increasing the buoyant density of the recombinant host cells or the density of the inclusion bodies produced by recombinant host cells should reduce the cost of isolating the polypeptide of interest. As such, an additional problem to be solved is to provide recombinant microbial host cells having one or more genetic modifications that increase the buoyant density of the cell or inclusion bodies produced within recombinant host cell and methods of using such modified microbial host cells to produce a polypeptide of interest.